AffinityImmuno Products

Introduction

ROBO4 is a member of the Roundabout receptor family expressed almost exclusively on vascular endothelial cells. It binds Slit ligands, most notably SLIT2, and this interaction restrains endothelial cell migration while helping to organize new blood vessel growth. A separate, largely Slit-independent action of ROBO4 helps hold endothelial cell-cell junctions together, preserving the integrity of the vascular barrier and limiting vessel leakiness. Altered ROBO4 signaling has been linked to bicuspid aortic valve disease, and shifts in its circulating levels have been explored as indicators of vascular stress in conditions such as pre-eclampsia.

Because ROBO4 helps maintain endothelial cell-cell junctions and limit vessel leakiness, shifts in its circulating levels are being explored as indicators of vascular stress in preeclampsia, giving this assay relevance to obstetric and cardiovascular vascular-biology research beyond its role in bicuspid aortic valve disease.

Principle of the Assay

This kit uses a sandwich enzyme immunoassay format in which the microtiter plate wells are pre-coated with a capture antibody specific to Human ROBO4. Standards or samples are added to the appropriate wells together with a biotin-conjugated detection antibody that also recognizes Human ROBO4. Avidin conjugated to horseradish peroxidase (HRP) is then added to each well and allowed to incubate. Upon addition of TMB substrate solution, a colorimetric signal develops exclusively in wells where Human ROBO4, the biotin-conjugated detection antibody, and avidin-HRP are all present. The enzymatic reaction is stopped by the addition of a sulphuric acid stop solution, and absorbance is measured at 450 nm (± 10 nm). Human ROBO4 concentrations in the samples are determined by comparing sample optical density values against a standard curve.